Isolation of a dual COX-2 and 5-lipoxygenase inhibitor from Acacia

ABSTRACT

The present invention provides a novel method for simultaneously inhibiting the cyclooxygenase COX-2 and 5-lipoxygenase (5-LO) enzymes. The method for the simultaneous dual inhibition of COX-2 and 5-LO is comprised of administering a composition containing an individual and/or a mixture of multiple flavans isolated from a single plant or multiple plants in the Acacia genus of plants to a host in need thereof. The present also includes novel methods for the prevention and treatment of COX-2 and 5-LO mediated diseases and conditions. The method for preventing and treating COX-2 and 5-LO mediated diseases and conditions is comprised of administering to a host in need thereof an effective amount of a composition comprising an individual and/or a mixture of multiple flavans isolated from a single plant or multiple plants in the Acacia genus of plants and a pharmaceutically acceptable carrier. The present invention includes a method for isolating and purifying a composition of flavans having dual specificity for COX-2 and 5-LO from the Acacia genus of plants.

FIELD OF THE INVENTION

[0001] This invention relates generally to a method for the prevention and treatment diseases and conditions mediated by COX-2 and 5-lipoxygenase. Specifically, the present invention relates to a method for the prevention and treatment of COX-2 and 5-lipoxygenase mediated diseases and conditions by administration of a class of specific compounds, referred to as flavans, extracted from Acacia plants. Included in this invention is a method to generate standardized flavan extracts from plant sources.

BACKGROUND OF THE INVENTION

[0002] The liberation and metabolism of arachidonic acid (AA) from the cell membrane, results in the generation of pro-inflammatory metabolites by several different pathways. Arguably, two of the most important pathways to inflammation are mediated by the enzymes 5-lipoxygenase (5-LO) and cyclooxygenase (COX). These are parallel pathways that result in the generation of leukotrienes and prostaglandins, respectively, which play important roles in the initiation and progression of the inflammatory response. These vasoactive compounds are chemotaxins, which both promote infiltration of inflammatory cells into tissues and serve to prolong the inflammatory response. Consequently, the enzymes responsible for generating these mediators of inflammation have become the targets for many new drugs aimed at the treatment of inflammation, which that contributes to the pathogenesis of diseases such as rheumatoid arthritis, osteoarthritis, Alzheimer's disease and certain types of cancer.

[0003] Inhibition of the enzyme cyclooxygenase (COX) is the mechanism of action attributed to most nonsteroidal anti-inflammatory drugs (NSAIDS). There are two distinct isoforms of the COX enzyme (COX-1 and COX-2) that share approximately 60% sequence homology, but differ in expression profiles and function. COX-1 is a constitutive form of the enzyme that has been linked to the production of physiologically important prostaglandins, which help regulate normal physiological functions, such as platelet aggregation, protection of cell function in the stomach and maintenance of normal kidney function. (Dannhardt and Kiefer (2001) Eur. J. Med. Chem. 36:109-26). The second isoform, COX-2, is a form of the enzyme that is inducible by pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and other growth factors. (Herschmann (1994) Cancer Metastasis Rev. 134:241-56; Xie et al. (1992) Drugs Dev. Res. 25:249-65). This isoform catalyzes the production of prostaglandin E2 (PGE2) from arachidonic acid (AA). Inhibition of COX-2 is responsible for the anti-inflammatory activities of conventional NSAIDs.

[0004] Inhibitors that demonstrate dual specificity for COX-2 and 5-LO while maintaining COX-2 selectivity relative to COX-1 would have the obvious benefit of inhibiting multiple pathways of arachidonic acid metabolism. Such inhibitors would block the inflammatory effects of PGE2, as well as, those of multiple leukotrienes (LT) by limiting their production. This includes the vasodilation, vasopermeability and chemotactic effects of LTB4 and LTD4 and the effects of LTE4, also known as the slow reacting substance of anaphalaxis. Of these, LTB4 has the most potent chemotactic and chemokinetic effects (Moore (1985) Prostanoids: pharmacological, physiological and clinical relevance. Cambridge University Press, N.Y., pp. 229-30) and has been shown to be elevated in the gastrointestinal mucosa of patients with inflammatory bowel disease (Sharon and Stenson (1983) Gastroenterology 84:1306-13) and within the synovial fluid of patients with rheumatoid arthritis. (Klicksein et al. (1980) J. Clin. Invest. 66:1166-70; Rae et al. (1982) Lancet ii:1122-4).

[0005] In addition to the above-mentioned benefits of dual COX-2/5-LO inhibitors, many dual inhibitors do not cause some of the side effects that are typical of NSAIDs or COX-2 inhibitors, including both the gastrointestinal damage and discomfort caused by traditional NSAIDs. It has been suggested that NSAID induced gastric inflammation is largely due to metabolites of 5-LO, particularly LTB4. (Kircher et al. (1997) Prostaglandins leukotrienes and essential fatty acids 56:417-23). Leukotrienes represent the primary arachidonic acid metabolites within the gastric mucosa following prostanoid inhibition. It appears that these compounds contribute to a significant amount of the gastric epithelial injury resulting from the use of NSAIDs. (Celotti and Laufer (2001) Pharmacological Research 43:429-36). Dual inhibitors of COX and 5-LO were also demonstrated to inhibit the coronary vasoconstriction in arthritic hearts in a rat model. (Gok et al. (2000) Pharmacology 60:41-46). Taken together, these characteristics suggest that there may be distinct advantages to dual inhibitors of COX-2 and 5-LO over COX-2 inhibitors and NSAIDs alone with regard to both increased efficacy and a lack of side effects.

[0006] Because the mechanism of action of COX inhibitors overlaps that of most conventional NSAID's, COX inhibitors are used to treat many of the same symptoms, including pain and swelling associated with inflammation in transient conditions and chronic diseases in which inflammation plays a critical role. Transient conditions include treatment of inflammation associated with minor abrasions, sunburn or contact dermatitis, as well as, the relief of pain associated with tension and migraine headaches and menstrual cramps. Applications to chronic conditions include arthritic diseases, such as rheumatoid arthritis and osteoarthritis. Although, rheumatoid arthritis is largely an autoimmune disease and osteoarthritis is caused by the degradation of cartilage in joints, reducing the inflammation associated with each provides a significant increase in the quality of life for those suffering from these diseases. (Wienberg (2001) Immunol. Res. 22:319-41; Wollhiem (2000) Curr. Opin. Rheum. 13:193-201). In addition to rheumatoid arthritis, inflammation is a component of rheumatic diseases in general. Therefore, the use of COX inhibitors has been expanded to include diseases, such as systemic lupus erythromatosus (SLE) (Goebel et al. (1999) Chem. Res. Tox. 12:488-500; Patrono et al. (1985) J. Clin. Invest. 76:1011-1018), as well as, rheumatic skin conditions, such as scleroderma. COX inhibitors are also used for the relief of inflammatory skin conditions that are not of rheumatic origin, such as psoriasis, in which reducing the inflammation resulting from the over production of prostaglandins could provide a direct benefit. (Fogh et al. (1993) Acta Derm Venerologica 73:191-3). Simply stated COX inhibitors are useful for the treatment of symptoms of chronic inflammatory diseases, as well as, the occasional ache and pain resulting from transient inflammation.

[0007] In addition to their use as anti-inflammatory agents, another potential role for COX inhibitors is in the treatment of cancer. Over expression of COX-2 has been demonstrated in various human malignancies and inhibitors of COX-2 have been shown to be efficacious in the treatment of animals with skin, breast and bladder tumors. While the mechanism of action is not completely understood, the over expression of COX-2 has been shown to inhibit apoptosis and increase the invasiveness of tumorgenic cell types. (Dempke et al. (2001) J. Can. Res. Clin. Oncol. 127:411-17; Moore and Simmons (2000) Current Med. Chem. 7:1131-44). It is possible that enhanced production of prostaglandins resulting from the over expression of COX-2 promotes cellular proliferation and consequently, increases angiogenesis. (Moore (1985) in Prostanoids: pharmacological, physiological and clinical relevance, Cambridge University Press, N.Y., pp. 229-30; Fenton et al. (2001) Am. J. Clin. Oncol. 24:453-57).

[0008] There have been a number of clinical studies evaluating COX-2 inhibitors for potential use in the prevention and treatment of different types of cancer. In 1999, 130,000 new cases of colorectal cancer were diagnosed in the U.S. Aspirin, a non-specific NSAID, for example, has been found to reduce the incidence of colorectal cancer by 40-50% (Giovannucci et al. (1995) N Engl J Med. 333:609-614) and mortality by 50% (Smalley et al. (1999) Arch Intern Med. 159:161-166). In 1999, the FDA approved the COX-2 inhibitor CeleCOXib for use in FAP (Familial Ademonatous Polyposis) to reduce colorectal cancer mortality. It is believed that other cancers, with evidence of COX-2 involvement, may be successfully prevented and/or treated with COX-2 inhibitors including, but not limited to esophageal cancer, head and neck cancer, breast cancer, bladder cancer, cervical cancer, prostate cancer, hepatocellular carcinoma and non-small cell lung cancer. (Jaeckel et al. (2001) Arch. Otolarnygol. 127:1253-59; Kirschenbaum et al. (2001) Urology 58:127-31; Dannhardt and Kiefer (2001) Eur. J. Med. Chem. 36:109-26). COX-2 inhibitors may also prove successful in preventing colon cancer in high-risk patients. There is also evidence that COX-2 inhibitors can prevent or even reverse several types of life-threatening cancers. To date, as many as fifty studies show that COX-2 inhibitors can prevent premalignant and malignant tumors in animals, and possibly prevent bladder, esophageal and skin cancers as well. COX-2 inhibition could prove to be one of the most important preventive medical accomplishments of the century.

[0009] Recent scientific progress has identified correlations between COX-2 expression, general inflammation and the pathogenesis of Alzheimer's disease (AD). (Ho et al. (2001) Arch. Neurol. 58:487-92). In animal models, transgenic mice that over express the COX-2 enzyme have neurons that are more susceptible to damage. The National Institute on Aging (NIA) is launching a clinical trial to determine whether NSAIDs can slow the progression of Alzheimer's disease. Naproxen (a non-selective NSAID) and rofecoxib (Vioxx, a COX-2 specific selective NSAID) will be evaluated. Previous evidence has indicated inflammation contributes to Alzheimer's disease. According to the Alzheimer's Association and the NIA, about 4 million people suffer from AD in the U.S.; and this is expected to increase to 14 million by mid-century.

[0010] The COX enzyme (also known as prostaglandin H2 synthase) catalyzes two separate reactions. In the first reaction, arachidonic acid is metabolized to form the unstable prostaglandin G2 (PGG2), a cyclooxygenase reaction. In the second reaction, PGG2 is converted to the endoperoxide PGH2, a peroxidase reaction. The short-lived PGH2 non-enzymatically degrades to PGE2. The compounds described herein are the result of a discovery strategy that combined an assay focused on the inhibition of COX-1 and COX-2 peroxidase activity with a chemical dereplication process to identify novel inhibitors of the COX enzymes.

[0011] Acacia is a genus of leguminous trees and shrubs. The genus Acacia includes more than 1000 species belonging to the family of Leguminosae and the subfamily of Mimosoideae. Acacias are distributed worldwide in tropical and subtropical areas of central and south America, Africa, parts of Asia, as well as, Australia, which has the largest number of endemic species. Acacias occur primarily in dry and arid regions, where the forests are often in the nature of open thorny shrubs. The genus Acacia is divided into 3 subgenera based mainly on the leaf morphology—Acacia, Aculiferum and Heterophyllum. Based on the nature of the leaves of mature trees, however, the genus Acacia can be divided into two “popular” groups: the typical bipinnate leaved species and the phyllodenous species. A phyllode is a modified petiole expanded into a leaflike structure with no leaflets, an adaptation to xerophytic conditions. The typical bipinnate leaved species are found primarily throughout the tropics, whereas the phyllodenous species occur mainly in Australia. More than 40 species of Acacia have been reported in India. Gamble in his Flora of Madras Presidency listed 23 native species for southern India, 15 of which are found in Tamil Nadu. Since that time, many new Acacia species have been introduced to India. Approximately 40 species are now found in Tamil Nadu itself. The indigenous species are primarily thorny trees or shrubs and a few are thorny stragglers, such as A. caesia, A. pennata and A. sinuata. Many species have been introduced from Africa and Australia, i.e. Acacia mearnsii, A. picnantha and A. dealbata, which have bipinnate leaves and A. auriculiformis, A. holoserecia and A. mangium, which are phyllodenous species.

[0012] Acacias are very important economically, providing a source of tannins, gums, timber, fuel and fodder. Tannins, which are isolated primarily from bark, are used extensively for tanning hides and skins. Some Acacia barks are also used for flavoring local spirits. Some indigenous species like A. sinuata also yield saponins, which are any of various plant glucosides that form soapy lathers when mixed and agitated with water. Saponins are used in detergents, foaming agents and emulsifiers. The flowers of some Acacia species are fragrant and used to make perfume. For example, Cassie perfume is obtained from Acacia ferrugenea. The heartwood of many Acacias is used for making agricultural implements and also provides a source of firewood. Acacia gums find extensive use in medicine and confectionary and as sizing and finishing materials in the textile industry. Lac insects can be grown on several species, including A. nilotica and A. catechu. Some species have been used for forestation of wastelands, including A. nilotica, which can withstand some water inundation and a few such areas have become bird sanctuaries.

[0013] To date, approximately 330 compounds have been isolated from various Acacia species. Flavonoids, a type of water-soluble plant pigments, are the major class of compounds isolated from Acacias. Approximately 180 different flavonoids have been identified, 111 of which are flavans. Terpenoids are second largest class of compounds isolated from species of the Acacia genus, with 48 compounds having been identified. Other classes of compounds isolated from Acacia include, alkaloids (28), amino acids/peptides (20), tannins (16), carbohydrates (15), oxygen heterocycles (15) and aliphatic compounds (10). (Buckingham, The Combined Chemical Dictionary, Chapman & Hall CRC, version 5:2, Dec. 2001).

[0014] Phenolic compounds, particularly flavans are found in moderate to high concentrations in all Acacia species. (Abdulrazak et al. (2000) Journal of Animal Sciences. 13:935-940). Historically, most of the plants and extracts of the Acacia genus have been utilized as astringents to treat gastrointestinal disorders, diarrhea and indigestion and to stop bleeding. (Vautrin (1996) Universite Bourgogne (France) European abstract 58-01C:177; Saleem et al. (1998) Hamdard Midicus. 41:63-67). The bark and pods of Acacia arabica Willd contain large quantities of tannins and have been utilized as astringents and expectorants. (Nadkarni (1996) India Materia Medica, Bombay Popular Prakashan, pp. 9-17). Diarylpropanol derivatives, isolated from stem bark of Acacia tortilis from Somalia, have been reported to have smooth muscle relaxing effects. (Hagos et al. (1987) Planta Medica. 53:27-31, 1987). It has also been reported that terpenoid saponins isolated from Acacia victoriae have an inhibitory effect on dimethylbenz(a)anthracene-induced murine skin carcinogenesis (Hanausek et al. (2000) Proceedings American Association for Cancer Research Annual Meeting 41:663) and induce apotosis (Haridas et al. (2000) Proceedings American Association for Cancer Research Annual Meeting. 41:600). Plant extracts from Acacia nilotica have been reported to have spasmogenic, vasoconstrictor and anti-hypertensive activity (Amos et al. (1999) Phytotherapy Research 13:683-685; Gilani et al. (1999) Phytotherapy Research. 13:665-669), and antiplatelet aggregatory activity (Shah et al. (1997) General Pharmacology. 29:251-255). Anti-inflammatory activity has been reported for A. nilotica. It was speculated that flavonoids, polysaccharides and organic acids were potential active components. (Dafallah and Al-Mustafa (1996) American Journal of Chinese Medicine. 20 24:263-269). To date, the only reported 5-lipoxygenase inhibitor isolated from Acacia is a monoterpenoidal carboxamide (Seikine et al. (1997) Chemical and Pharmaceutical Bulletin. 45:148-11).

[0015] Acacia gums have been formulated with other plant ingredients and used for ulcer prevention without identification of any of the active components. (Fuisz, U.S. Pat. No. 5,651,987). Acacia gums have also been formulated with other plant ingredients and used to improve drug dissolution (Blank, U.S. Pat. No. 4,946,684), by lowering the viscosity of nutritional compositions (Chancellor, U.S. Pat. No. 5,545,411).

[0016] The extract from the bark of Acacia has been patented in Japan for external use as a whitening agent (Abe, JP10025238), as a glucosyl transferase inhibitor for dental applications (Abe, JP07242555), as a protein synthesis inhibitor (Fukai, JP 07165598), as an active oxygen scavenging agent for external skin preparations (Honda, JP 07017847, Bindra U.S. Pat. No. 6,1266,950), and as a hyaluronidase inhibitor for oral consumption to prevent inflammation, pollinosis and cough (Ogura, JP 07010768).

[0017] Catechin is a flavan, found primarily in green tea, having the following structure.

[0018] Catechin works both alone and in conjunction with other flavonoids found in tea, and has both antiviral and antioxidant activity. Catechin has been shown to be effective in the treatment of viral hepatitis. It also appears to prevent oxidative damage to the heart, kidney, lungs and spleen. Catechin also has been shown to inhibit the growth of stomach cancer cells.

[0019] Catechin and its isomer epicatechin inhibit prostaglandin endoperoxide synthase with an IC₅₀ value of 40 μmol/L. (Kalkbrenner et al. (1992) Pharmacol. 44:1-12). Five flavan-3-ol derivatives, including (+)-catechin and gallocatechin, isolated from four plant species: Atuna racemosa, Syzygium carynocarpum, Syzygium malaccense and Vantanea peruviana, exhibit equal to weaker inhibitory activity against COX-2, relative to COX-1, with IC₅₀ values ranging from 3.3 μM to 138 μM (Noreen et al. (1998) Planta Med. 64:520-524). (+)-Catechin, isolated from the bark of Ceiba pentandra, inhibits COX-1 with an IC₅₀ value of 80 μM (Noreen et al. (1998) J. Nat. Prod. 61:8-12). Commercially available pure (+)-catechin inhibits COX-1 with an IC₅₀ value of around 183 to 279 μM depending upon the experimental conditions, with no selectivity for COX-2. (Noreen et al. (1998) J. Nat. Prod. 61:1-7).

[0020] Green tea catechin, when supplemented into the diets of Dawley male rats, lowered the activity level of platelet phospholipase A2 and significantly reduced platelet cyclooxygenase levels. (Yang et al. (1999) J. Nutr. Sci. Vitaminol. 45:337-346). Catechin and epicatechin reportedly weakly suppress COX-2 gene transcription in human colon cancer DLD-1 cells (IC₅₀=415.3 μM). (Mutoh et al. (2000) Jpn. J. Cancer Res. 91:686-691). The neuroprotective ability of (+)-catechin from red wine results from the antioxidant properties of catechin, rather than inhibitory effects on intracellular enzymes, such as cyclooxygenase, lipoxygenase, or mitric oxide synthase (Bastianetto et al. (2000) Br. J. Pharmacol. 131:711-720). Catechin derivatives purified from green tea and black tea, such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and theaflavins showed inhibition of cyclooxygenase and lipoxygenase dependent metabolism of arachidonic acid in human colon mucosa and colon tumor tissues (Hong et al. (2001) Biochem. Pharmacol. 62:1175-1183) and induce COX-2 expression and PGE(2) production (Park et al. (2001) Biochem. Biophys. Res. Commun. 286:721-725). Epiafzelechin isolated from the aerial parts of Celastrus orbiculatus exhibited a dose-dependent inhibition of COX-1 activity with an IC₅₀ value of 15 μM and also demonstrated anti-inflammatory activity against carrageenin-induced mouse paw edema following oral administration at a dosage of 100 mg/kg. (Min et al. (1999) Planta Med. 65:460-462).

[0021] Catechin and its derivatives from various plant sources, especially from green tea leaves, have been used in the treatment of HPV infected Condyloma acuminata (Cheng, U.S. Pat. No. 5,795,911) and in the treatment of hyperplasia caused by papilloma virus (Cheng, U.S. Pat. Nos. 5,968,973 and 6,197,808). Catechin and its derivatives have also been used topically to inhibit angiogenesis in mammalian tissue, such as skin cancer, psoriasis, spider veins or under eye circles (Anderson, U.S. Pat. No. 6,248,341), against UVB-induced tumorigenesis on mice (Agarwal et al. (1993) Photochem. Photobiol. 58:695-700), for inhibiting nitric oxide synthase at the level of gene expression and enzyme activity (Chan, U.S. Pat. No. 5,922,756), as a hair-growing agent (Takahashi, U.S. Pat. No. 6,126,940). Catechin based compositions have also been formulated with other extracts and vitamins for treatment of acne (Murad U.S. Pat. No. 5,962,517), hardening the tissue of digestive organs (Shi, U.S. Pat. No. 5,470,589), for inhibiting 5 alpha-reductase activity in treating androgenic disorder related diseases and cancers (Liao, U.S. Pat. No. 5,605,929). Green tea extract has been formulated with seven other plant extracts for reducing inflammation by inhibiting the COX-2 enzyme, without identification of any of the specific active components (Mewmark, U.S. Pat. No. 6,264,995).

[0022] To date, a number of naturally occurring flavonoids have been commercialized for varying uses. For example, liposome formulations of Scutellaria extracts have been utilized for skin care (U.S. Pat. Nos. 5,643,598; 5,443,983). Baicalin has been used for preventing cancer, due to its inhibitory effects on oncogenes (U.S. Pat. No. 6,290,995). Baicalin and other compounds have been used as antiviral, antibacterial and immunomodulating agents (U.S. Pat. No. 6,083,921) and as natural anti-oxidants (Poland Pub. No. 9,849,256). Chrysin has been used for its anxiety reducing properties (U.S. Pat. No. 5,756,538). Anti-inflammatory flavonoids are used for the control and treatment of anorectal and colonic diseases (U.S. Pat. No. 5,858,371), and inhibition of lipoxygenase (U.S. Pat. No. 6,217,875). These compounds are also formulated with glucosamine collagen and other ingredients for repair and maintenance of connective tissue (Bath, U.S. Pat. No. 6,333,304). Flavonoid esters constitute active ingredients for cosmetic compositions (U.S. Pat. No. 6,235,294). The bark, extract and compounds derived from Phellodendron amurense have been patented for use in treatment of inflammatory diseases (U.S. Pat. Nos. 5,766,614; 5,908,628; 6,113,909; 6,193,977). Cherry bioflavonoids from Prunus avium and Prunus cerasus with anthocyanidin type of structures have been patented as cyclooxygenase inhibitors (U.S. Pat. No. 6,194,469, U.S. patent application 20010002407).

SUMMARY OF THE INVENTION

[0023] The present invention includes methods that are effective in simultaneously inhibiting the enzymes COX-2 and 5-lipoxygenase. The method for simultaneous dual inhibition of the enzymes COX-2 and 5-LO is comprised of administering a composition comprising an individual and/or a mixture of multiple flavans isolated from a single plant or multiple plants in the Acacia genus of plants to a host in need thereof.

[0024] The present also includes methods for the prevention and treatment of COX-2 and 5-LO mediated diseases and conditions. The method for preventing and treating COX-2 and 5-LO mediated diseases and conditions is comprised of administering to a host in need thereof an effective amount of a composition comprising an individual and/or a mixture of multiple flavans isolated from a single plant or multiple plants in the Acacia genus of plants and a pharmaceutically acceptable carrier.

[0025] The flavans that can be used in accordance with the following include compounds illustrated by the following general structure:

[0026] wherein

[0027] R₁, R₂, R₃, R₄ and R₅ are independently selected from the group consisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters of the mentioned substitution groups, including, but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; thereof carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, but not limited to, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized flavans;

[0028] wherein

[0029] R is an alkyl group having between 1-10 carbon atoms; and

[0030] X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.

[0031] The method of this invention can be used to treat and prevent a number of COX-2 and 5-LO mediated diseases and conditions including, but not limited to, osteoarthritis, rheumatoid arthritis, menstrual cramps, systemic lupus erythromatosus, psoriasis, chronic tension headaches, migraine headaches, topical wound and minor inflammatory conditions, inflammatory bowel disease and solid cancers.

[0032] As noted above the flavans of this invention may be obtained from a plant or plants selected from the genus of Acacia. In a preferred embodiment, the plant is selected from the group consisting of Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.

[0033] The compositions of this invention can be administered by any method known to one of ordinary skill in the art. The modes of administration include, but are not limited to, enteral (oral) administration, parenteral (intravenous, subcutaneous, and intramuscular) administration and topical application. The method of treatment according to this invention comprises administering internally or topically to a patient in need thereof a therapeutically effective amount of the individual and/or a mixture of multiple flavans from a single source or multiple sources that include, but not limited to, synthetically obtained, naturally occurring, or any combination thereof.

[0034] The present invention includes a method for isolating and purifying flavans from the Acacia genus of plants. The method of the present invention comprises: a) extracting the ground biomass of a plant selected from the Acacia genus of plants; b) neutralizing and concentrating said extract; and c) purifying said neutralized and concentrated extract. In a preferred embodiment of the invention the extract is purified using a method selected from the group consisting of recrystallization, precipitation, solvent partition and/or chromatographic separation. The present invention provides a commercially viable process for the isolation and purification of Acacia flavans having desirable physiological activity.

[0035] The present invention implements a strategy that combines a series of biomolecular screens with a chemical dereplication process to identify active plant extracts and the particular compounds within those extracts that specifically inhibit COX-2 and 5-LO enzymatic activity and inflammation. A total of 1230 plant extracts were screened for their ability to inhibit the peroxidase activity associated with recombinant COX-2. This primary screen identified 22 plant extracts that were further studied for their ability to specifically and selectively inhibit COX-2 in vitro in both cell based and whole blood assays. Those extracts that were efficacious in vitro were then tested for their ability to inhibit inflammation in vivo using a both air pouch and topical ear-swelling models of inflammation when administered by multiple routes (IP and oral). These studies resulted in the discovery of botanical extracts that inhibited COX-2 activity and were efficacious both in vitro and in vivo. These extracts were then tested for their ability to inhibit 5-LO. These studies resulted in the identification of flavan extracts from the Acacia genus of plants that demonstrate dual specificity for COX-2 and 5-LO. Applicant believes that this is first report of a composition of matter isolated from the Acacia genus of plants that demonstrates this dual specificity for COX-2 and 5-LO.

[0036] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1 depicts graphically a profile of the inhibition of COX-1 and COX-2 by HTP fractions from organic extracts of Acacia catechu. The extracts were examined for their inhibition of the peroxidase activity of recombinant ovine COX-1 (▪) or ovine COX-2 (). The data is presented as percent of untreated control.

[0038]FIG. 2 depicts graphically a profile of the inhibition of COX-1 and COX-2 by HTP fractions from aqueous extracts of Acacia catechu. The extracts were examined for their inhibition of the peroxidase activity of recombinant ovine COX-1 (▪) or ovine COX-2 (). The data is presented as percent of untreated control.

[0039]FIG. 3 depicts the results of LC/PDA/MS analysis of active HTP fraction D11, which is described in Example 3. FIG. 3A depicts the LC/PDA chromatogram of HTP fraction D11. FIG. 3B depicts the selected ion chromatogram of HTP fraction D11 at m/z=579 and the structure of the compound robinetinidol-(4-6/8)-catechin. FIG. 3C depicts the selected ion chromatogram of HTP fraction D11 at m/z=291 and the structure of the compound catechin.

[0040]FIG. 4 depicts the high-pressure liquid chromatography (HPLC) traces of the organic (FIG. 4A) and aqueous (FIG. 4B) extracts from Acacia catechu.

[0041]FIG. 5 illustrates graphically the effect of Acacia catechu organic extracts on 5-lipoxygenase activity.

[0042]FIG. 6 illustrates the inhibition of arachidonic acid induced inflammation by Acacia catechu. The in vivo efficacy was evaluated based on the ability to inhibit swelling induced by direct application of arachidonic acid as described in Example 9. The average difference in swelling between treated ears and control ears is depicted in FIG. 6A. FIG. 6B depicts the percent inhibition of each group in comparison to the arachidonic acid treated control.

[0043]FIG. 7 illustrates the effect of standardized Acacia extracts on Zymosan induced inflammation. Zymosan was used to elicit a pro-inflammatory response in an air pouch as described in Example 9. Markers of inflammation including infiltration of pro-inflammatory cells (FIG. 7A), MPO concentrations under different experimental conditions (FIG. 7B) and percent inhibition of MPO activity within the air pouch fluid (FIG. 7C) were used to evaluate the efficacy and mechanism of action of the anti-inflammatory activity of Acacia extracts.

[0044]FIG. 8 depicts the HPLC tract of the flavans extracted from Acacia catechu with 80% MeOH in water.

DETAILED DESCRIPTION OF THE INVENTION

[0045] Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided.

[0046] “Flavans” are a specific class of flavonoids, which can be generally represented by the following general structure:

[0047] wherein

[0048] R₁, R₂, R₃, R₄ and R₅ are independently selected from the group consisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters of substitution groups, including, but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; thereof carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, but not limited to, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized flavans;

[0049] wherein

[0050] R is an alkyl group having between 1-10 carbon atoms; and

[0051] X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.

[0052] “Therapeutic” as used herein, includes treatment and/or prophylaxis. When used, therapeutic refers to humans, as well as, other animals.

[0053] “Pharmaceutically or therapeutically effective dose or amount” refers to a dosage level sufficient to induce a desired biological result. That result may be the delivery of a pharmaceutical agent, alleviation of the signs, symptoms or causes of a disease or any other desired alteration of a biological system.

[0054] A “host” is a living subject, human or animal, into which the compositions described herein are administered.

[0055] Note, that throughout this application various citations are provided. Each citation is specifically incorporated herein in its entirety by reference.

[0056] The present invention includes methods that are effective in simultaneously inhibiting both the cyclooxygenase (COX-2) and 5-lipoxygenase (5-LO) enzymes. The method for the simultaneous dual inhibition of the COX-2 and 5-LO enzymes is comprised of administering a composition comprising an individual and/or a mixture of multiple flavans isolated from a single plant or multiple plants selected from the Acacia genus of plants to a host in need thereof.

[0057] The present also includes methods for the prevention and treatment of COX-2 and 5-LO mediated diseases and conditions. The method for preventing and treating COX-2 and 5-LO mediated diseases and conditions is comprised of administering to a host in need thereof an effective amount of a composition comprising an individual and/or a mixture of multiple flavans isolated from a single plant or multiple plants selected from the Acacia genus of plants and a pharmaceutically acceptable carrier.

[0058] The flavans that can be used in accordance with the method of this invention include compounds illustrated by the general structure set forth above. The flavans of this invention are isolated from a plant or plants selected from the Acacia genus of plants. In a preferred embodiment, the plant is selected from the group consisting of Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.

[0059] The flavans can be found in different parts of plants, including but not limited to stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts.

[0060] In order to identify compounds able to inhibit the COX enzymes an extract library composed of 1230 extracts from 615 medicinal plants collected from China, India, and other countries was created. This primary screen identified 22 plant extracts that were further studied for their ability to specifically and selectively inhibit COX-2 in vitro in both cell based and whole blood assays. A general method for preparing the extracts is described in Example 1, which uses the Acacia catechu species for purposes of illustration. The ground powder of the bark from Acacia catechu was extracted with an organic solvent followed by deionized (DI) water. The extraction process yields an organic and an aqueous extract for each species examined. The results of the extraction are set forth in Table 1. These primary extracts are the source material used in the preliminary assay to identify inhibitors of the cyclooxygenase enzyme's peroxidase activity, which is one of the main functional activities of cyclooxygenase and is responsible for converting PGG2 to PGH2 and ultimately PGE2, as described in detail above. This assay is described in Example 2 and the results are set forth in Table 2. With reference to Table 2, it can be seen that the organic and aqueous extracts from Acacia catechu inhibited the peroxidase activity of COX-2 albeit to differing degrees.

[0061] The COX-2 inhibitory activity from the primary assay of the crude extracts was confirmed by measurement of dose response and IC₅₀ (the concentration required to inhibit 50% of the enzyme's activity). The IC₅₀ values are set forth in Table 3. In this assay, Acacia catechu organic extract was efficacious (IC₅₀=3/6 μg/mL) against human/ovine COX-2 and ovine COX-1 (IC₅₀=2.5 μg/mL). Thus, the primary screens for inhibitors of the COX enzyme revealed that the organic extract from Acacia genus was efficacious against COX-2 enzyme.

[0062] In order to efficiently identify active compounds from plant extracts, a high throughput fractionation process was used, as described in Example 3. Briefly, the organic and aqueous extracts from Acacia catechu were fractionated with a high throughput purification system coupled with a due channel broadband UV detector and two Gilson 222XL liquid handlers. Eighty-eight fractions were collected in two 96 deep well plates. Each of the fractions was tested for its ability to inhibit COX activity as per the primary assay, as described in Example 4 and structure dereplication was initialized for the positive HTP fractions with off-line LC/PDA and LC/MS analyses. The results are set forth in FIGS. 1 and 2, which depict the profile of COX-1 and COX-2 inhibition by various HTP fractions derived from the organic and aqueous extracts of Acacia catechu, respectively. It should be noted that a number of the HTP fractions actually exhibit selective COX-2 inhibitory activity, suggesting that there are multiple compounds in the extracts that contribute to the observed selective inhibitory effects of the whole extract.

[0063] The active HTP fractions from the aqueous extracts labeled well #C8 to F7 were analyzed using LC/MS with positive mode. The LC/PDA/PDA/MS analysis of fraction D11 are set forth in FIG. 3. The first active peak was located between fractions C9 to D9 and contained catechin components with a [molecular ion +1]⁺ at 291 m/z. The second active peak, which exhibited COX-1 selectivity, was located between fractions D9 to E2 and contained catechin dimer, having a molecular ion at 578 m/z. The third active peak, which exhibited COX-2 selectivity, was located between fractions #E8 to F8 and contained multiple components with an m/z at 573 & 579, which corresponds to 4-hydroxycinnamoyl-oleanen-3-ol and robinetinidol-catechin, respectively.

[0064] The separation, purification and identification of the active components present in the organic extract of Acacia catechu is described in Example 5. Using the methodology described in Example 5, catechin and epicatechin were identified as the two major active compounds in the organic extract from the roots of Acacia catechu, having IC₅₀values of 5-7 μg/mL.

[0065] HPLC quantification of the active extracts from Acacia catechu is described in Example 6. The results are set forth in Table 4 which shows that the flavan content in the organic and aqueous extracts, as determined by HPLC, is 30.4% and 1.0%, respectively. This explains why the inhibitory activity of the organic extract is more than twice that of the aqueous extract. HPLC analysis also demonstrates that each extract contains minor flavan components, which contribute to the selective COX-2 inhibitory activity. The HPLC results are set forth in FIGS. 4A and B.

[0066] The primary assay described in Example 2 is a cell free system utilizing recombinant enzymes. To further demonstrate the biological activity of the active extracts and compounds, two models that represent cell based in vitro efficacy and animal based in vivo efficacy were employed. The method used to evaluate in vitro efficacy and selectivity is described in Example 7. Two cell lines were selected that could be induced to express primarily COX-1 (THP-1 cells) and COX-2 (HOSC cells), respectively. Each cell type was examined for the production of PGE₂, the primary product of the COX enzymes. The results are set forth in Table 5, which shows that the Acacia organic extract inhibits both the COX-1 and COX-2 enzymes with a preference for the COX-1 enzyme. While the use of the THP-1 cell line is important and demonstrates the ability of the active compounds to cross the cell membrane, it is an immortalized cell line therefore evaluation of efficacy based on a more relevant model system is desirable. As a result, the extract was also evaluated using whole blood as the primary source of both COX-1 and COX-2 activity. This system measures the inhibition of the production of PGE₂ vs. TXB₂ to differentiate between COX-2 and COX-1 inhibitory activity, respectively. The results, which are set forth in Table 5, demonstrate that both the COX-1 and COX-2 enzymes are inhibited by the Acacia extracts. The IC₅₀ values suggest that within this system the Acacia extracts are more efficacious against COX-2. Taken as a whole, the inhibitory effect of the active compounds within these extracts is significant and efficacious in both cell free and cell-based systems in vitro.

[0067] As noted above, dual inhibitors of both the COX and 5-LO enzymes have distinct advantages over COX inhibitors alone. These advantages include, but are not limited to, a broader effect on inhibition of arachidonic acid metabolites, a lack of gastrointestinal toxicity and a potential decrease in vasoconstriction. Therefore, the Acacia organic extracts were tested for their effects on 5-LO inhibition, as described in Example 8. Briefly, recombinant, human 5-LO was incubated with the Acacia extract for 15 minutes prior to the addition of enzyme substrate (umbelliferyl arachidonate). The results are set forth in FIG. 5. The Acacia extracts inhibited 5-LO activity in this cell free in vitro system and demonstrated an IC₅₀ of 70 μg/mL. Although the IC₅₀ of the Acacia extracts for 5-LO is higher than that of the same extract for COX-2, it potentially adds a significant benefit for treating inflammation induced by arachidonic acid metabolism.

[0068] Two separate in vivo models were employed to determine whether the in vitro efficacy observed from the Acacia extracts translated to an ability to inhibit in vivo inflammatory responses. The two models are described in Example 9. The first of these systems is designed to measure inflammation resulting directly from the arachidonic acid metabolism pathway. In this example, mice were treated with extracts from Acacia prior to the topical application of AA to the ear to induce the inflammatory response. The effect of pretreating the animals was then measured by the inhibition of the ear swelling using a micrometer. The Acacia extracts demonstrated a good inhibitory response suggesting specific inhibition of AA induced inflammatory responses. The results are set forth in FIGS. 6A and 6B. Additionally, the extracts also showed significant inhibition when administered either orally or interperitoneally. Thus, the Acacia extracts were demonstrated to be efficacious in reducing inflammation by multiple routes of administration.

[0069] The organic extract from Acacia was the most efficacious against inflammation induced directly by the arachidonic acid. Therefore, the efficacy of a standardized Acacia extract was examined using a second model in which multiple mechanisms of inflammation are ultimately responsible for the final effect. This system is therefore more relevant to naturally occurring inflammatory responses. Using this model, a very potent activator of the complement system is injected into an air pouch created on the back of Balb/C mice. This results in a cascade of inflammatory events including, infiltration of inflammatory cells, activation of COX enzymes, resulting in the release of PGE₂, the enzyme myeloperoxidase (MPO), and production of a very specific profile of pro-inflammatory cytokines including TNF-α. These studies demonstrated that even though the Acacia did not inhibit the initial infiltration (chemotactic response) of inflammatory cells into the air pouch, it blocked the activation of those cells. This is evidenced by the lack of MPO excreted into the extracellular fluid of the pouch and the noted lack of production of TNF-α. The results are set forth in FIG. 7. The data demonstrates that the Acacia extract is efficacious and helps control an inflammatory response in a model system where multiple inflammatory pathways are active. However, the standardized extract produced a toxic effect that was lethal to 60% of the animals receiving the highest concentration of Acacia extracts IP (200 mg/kg). This toxic effect was not observed in animals receiving the same dose by gavage.

[0070] The preparation of products for administration in pharmaceutical preparations may be performed by a variety of methods well known to those skilled in the art. The flavans may be formulated as an herb powder in the form of its natural existence; as solvent and/or supercritical fluid extracts in different concentrations; as enriched and purified compounds through recrystallization, column separation, solvent partition, precipitation and other means, as a pure and/or a mixture containing substantially purified flavans prepared by synthetic methods.

[0071] Various delivery systems are known in the art and can be used to administer the therapeutic compositions of the invention, e.g., aqueous solution, encapsulation in liposomes, microparticles, and microcapsules.

[0072] Therapeutic compositions of the invention may be administered parenterally by injection, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iontophoresis or suppositories are also envisioned. One preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used. In one preferred embodiment, it is envisioned that the carrier and flavan(s) constitute a physiologically compatible, slow release formulation. The primary solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmacologically acceptable excipients for modifying or maintaining the stability, rate of dissolution, release or absorption of the ligand. Such excipients are those substances usually and customarily employed to formulate dosages for parental administration in either unit dose or multi-dose form.

[0073] Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder; or directly capsulated and/or tableted with other inert carriers for oral administration. Such formulations may be stored either in a ready to use form or requiring reconstitution immediately prior to administration. The manner of administering formulations containing the compositions for systemic delivery may be via oral, subcutaneous, intramuscular, intravenous, intranasal or vaginal or rectal suppository.

[0074] The amount of the composition which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder of condition, which can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness or advancement of the disease or condition, and should be decided according to the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curved derived from in vitro or animal model test systems. For example, an effective amount of the composition of the invention is readily determined by administering graded doses of the composition of the invention and observing the desired effect.

[0075] The method of treatment according to this invention comprises administering internally or topically to a patient in need thereof a therapeutically effective amount of the individual and/or a mixture of multiple flavans from a single source or multiple sources. The purity of the individual and/or a mixture of multiple flavans includes, but is not limited to 0.01% to 100%, depending on the methodology used to obtain the compound(s). In a preferred embodiment doses of the flavans and pharmaceutical compositions containing the same are an efficacious, nontoxic quantity generally selected from the range of 0.01 to 200 mg/kg of body weight. Persons skilled in the art using routine clinical testing are able to determine optimum doses for the particular ailment being treated.

[0076] This invention includes an improved method for isolating and purifying flavans from Acacia plants, which is described in Example 10. In Example 10, flavans from Acacia catechu were extracted with different solvent systems. The results are set forth in Table 6. The improved method of this invention comprises: extraction of the ground biomass of a plant containing flavans with an organic solvent or a combination of organic solvent(s) and/or water; neutralization and concentration of the neutralized extract; and purification of said extract by recrystallization and/or chromatography. It can be seen from Table 6, that 80% methanol in water is one of the preferred solvents for extraction of flavans from Acacia plants. As provided above, these flavans can be can be isolated from the Acacia genus of plants. The method of this invention can be extended to the isolation of these compounds from any plant source containing these compounds.

[0077] Additionally the flavans can be isolated from various parts of the plant including, but not limited to, the whole plant, stems, stem bark, twigs, tubers, flowers, fruit, roots, root barks, young shoots, seeds, rhizomes and aerial parts. In a preferred embodiment the flavans are isolated from the roots, reproductive organs or the whole plant.

[0078] The solvents that can be used for extraction of the ground biomass of the plant include, but are not limited to water, acidified water, water in combination with miscible hydroxylated organic solvent(s) including, but not limited to, methanol or ethanol and an mixture of alcohols with other organic solvent(s) such as THF, acetone, ethyl acetate etc. In one embodiment the extract is neutralized to a pH of 4.5-5.5 and then concentrated and dried to yield a powder. The flavans can then be purified by various methods including, but not limited to recrystallization, solvent partition, precipitation, sublimation, and/or chromatographic methods including, but not limited to, ion exchange chromatography, absorption chromatography, reverse phase chromatography, size exclusive chromatography, ultra-filtration or a combination of two.

[0079] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES Example 1

[0080] Preparation of Organic and Aqueous Extracts from Acacia catechu

[0081] Plant material from Acacia catechu (L) Willd was ground to a particle size no larger than 2 mm. Dried ground plant material (60 g) was then transferred to an Erlenmeyer flask and methanol:dichloromethane (1:1) (600 mL) was added. The mixture was shaken for one hour, filtered and the biomass was extracted again with methanol:dichloromethane (1:1) (600 mL). The organic extracts were combined and evaporated under vacuum to provide the organic extract (see Table 1 below). After organic extraction, the biomass was air dried and extracted once with ultra pure water (600 mL). The aqueous solution was filtered and freeze-dried to provide the aqueous extract. TABLE 1 Yield of Organic and Aqueous Extracts of Acacia catechu Plant Source Amount Organic Extract Aqueous Extract Acacia catechu 60 g 27.2 10.8

Example 2

[0082] Inhibition of COX-2 and COX-1 Peroxidase Activity by Plant Extracts from Acacia catechu

[0083] The bioassay directed screening process for the identification of specific COX-2 inhibitors was designed to assay the peroxidase activity of the enzyme as described below.

[0084] Peroxidase Assay. The assay to detect inhibitors of COX-2 was modified for a high throughput platform (Raz and Needleman (1990) J. Biol. Chem. 269:603-607). Briefly, recombinant ovine COX-2 (Cayman) in peroxidase buffer (100 mM, TBS, 5 mM EDTA, 1 μM Heme, 0.01 mg epinephrine, 0.094% phenol) was incubated with the extract (1:500 dilution) for 15 minutes. Quantablu (Pierce) substrate was added and allowed to develop for 45 minutes at 25° C. Luminescence was then read using a Wallac Victor 2 plate reader. The results are set forth in Table 2. The data in Table 2 is presented as the percent of peroxidase activity relative to the recombinant ovine COX-2 enzyme and substrate alone. The percent inhibition ranged from 30% (70% of control) for the aqueous extract to 75% for the organic extract. The data clearly demonstrates that the organic extract is the more efficacious in vitro. TABLE 2 Inhibition of COX-2 Peroxidase activity by Acacia catechu Inhibition of COX-2 Inhibition of COX-2 Plant Source by organic extract by aqueous extract Acacia catechu 75% 30%

[0085] Comparison of the relative inhibition of the COX-1 and COX-2 isoforms requires the generation of IC₅₀ values for each of these enzymes. The IC₅₀ is defined as the concentration at which 50% inhibition of enzyme activity in relation to the control is achieved by a particular inhibitor. In the instant case, IC₅₀ values were found to range from 3 to 6 μg/mL and 2.5 μg/mL for the COX-2 and COX-1 enzymes, respectively, as set forth in Table 3. TABLE 3 IC₅₀ Values for Human and Ovine COX-2 and COX-1 IC₅₀ Human COX-2 IC₅₀ Ovine COX-2 IC₅₀ Ovine COX-1 Plant Source (μg/mL) (μg/mL) (μg/mL) Acacia catechu 3 6.25 2.5

Example 3

[0086] HTP Fractionation of Active Extracts

[0087] The organic extract (400 mg) from Acacia catechu was loaded onto a prepacked flash column. (2 cm ID×8.2 cm, 10 g silica gel). The column was eluted using an Hitachi high throughput purification (HTP) system with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 30 minutes at a flow rate of 5 mL/min. The separation was monitored using a broadband wavelength UV detector and the fractions were collected in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. The sample plate was dried under low vacuum and centrifugation. DMSO (1.5 mL) was used to dissolve the samples in each cell and a portion (100 μL) was taken for the COX inhibition assay.

[0088] The aqueous extract (750 mg) from Acacia catechu was dissolved in water (5 mL), filtered through a 1 μm syringe filter and transferred to a 4 mL HPLC vial. The mixture was then injected by an autosampler onto a prepacked reverse phase column (C-18, 15 μm particle size, 2.5 cm ID×10 cm with precolumn insert). The column was eluted using an Hitachi high throughput purification (HTP) system with a gradient mobile phase of (A) water and (B) methanol from 100% A to 100% B in 20 minutes, followed by 100% methanol for 5 minutes at a flow rate of 10 mL/min. The separation was monitored using a broadband wavelength UV detector and the fractions were collected in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. The sample plate was freeze-dried. Ultra pure water (1.5 mL) was used to dissolve samples in each cell and a portion of 100 μL was taken for the COX inhibition assay. The active HTP fractions from well #C8 to F7 were analyzed with LC/MS/PDA at positive mode with super sonic ionization source. The results for active fraction D11 are set forth in FIG. 3.

Example 4

[0089] Inhibition of COX Peroxidase Activity by HTP Fractions from Acacia catechu

[0090] Individual organic extracts were further characterized by examining each of the HTP fractions for the ability to inhibit the peroxidase activity of both COX-1 and COX-2 recombinant enzymes. The results are set forth in FIGS. 1 and 2, which depicts the inhibition of COX-2 and COX-1 activity by HTP fractions from Acacia isolated as described in Example 1. The profile depicted in FIG. 1 shows a peak of inhibition that is very selective for COX-2 and multiple peaks that inhibit both enzymes with equal efficacy. However, both the COX-1 and COX-2 enzymes demonstrate multiple peaks of inhibition suggesting that there is more than one molecule contributing to the initial inhibition profiles.

Example 5

[0091] Isolation and Purification of the Active Compounds from the Organic Extract of Acacia catechu

[0092] The organic extract (5 g) from the roots of Acacia catechu, isolated as described in Example 1, was loaded onto prepacked flash column (120 g silica, 40 μm particle size 32-60 μm, 25 cm×4 cm) and eluted with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 60 minutes at a flow rate of 15 mL/min. The fractions were collected in test tubes at 10 mL/fraction. The solvent was evaporated under vacuum and the sample in each fraction was dissolved in DMSO (1 mL) and an aliquot of 20 μL was transferred to a 96 well shallow dish plate and tested for COX inhibitory activity. Based upon the COX assay results, active fractions #32 to #41 were combined and evaporated to yield 2.6 g of solid. Analysis by HPLC/PDA and LC/MS showed two major compounds with retention times of 15.8 and 16.1 minutes, respectively. The product was further purified on a C18 semi-preparatory column (25 cm×1 cm), loaded with 212.4 mg of product and eluted with a gradient mobile phase of (A) water and (B) acetonitrile (ACN), over a period of 60 minutes at a flow rate of 5 mL/minute. Eighty-eight fractions were collected and two active compounds were isolated. Compound 1 (11.5 mg) and Compound 2 (16.6 mg). Purity was determined by HPLC/PDA and LC/MS data by comparison with standards (catechin and epicatechin) and NMR data.

[0093] Compound 1. ¹³C NMR: δ ppm (DMSO-d6) 27.84 (C4), 66.27 (C3), 80.96 (C2), 93.78 (C9), 95.05 (C7), 99.00 (C5), 114.48 (C12), 115.01 (C15), 118.36 (C16), 130.55 (C11), 144.79 (C14), 155.31 (C6), 156.12 (C10), 156.41 (C8). ¹H NMR: δ ppm. (DMSO-d6) 9.150 (1H, s, OH), 8.911 (1H,s, OH), 8.835 (1H, s, OH), 8.788 (1H, s, OH), 6.706 (1H, d, J=2 Hz, H2′), 6.670 (1H, d, J=8.0 Hz, H-6′), 6.578 (1H, dd, J=2, 8 Hz, H-5′), 5.873 (1H, d, J=2 Hz, H8), 5.670 (1H, d, J=2 Hz, H6), 4.839 (1H, d, J=4 Hz, OH), 4.461 (1H, d, J=7.3 Hz, H2), 3.798 (1H, m, H3), 2.625 (1H, m, H4b), 2.490 (1H, m, H4a). MS: [M+1]⁺=291 m/e. This compound has been identified as catechin. The IC₅₀ values of epicatechin against the COX-1 and COX-2 enzymes are 6 μg/mL and 40 μg/mL, respectively.

[0094] Compound 2. ¹³C NMR: δ ppm. (DMSO-d6) 28.17 (C4), 64.87 (C3), 78.02 (C2), 94.03 (C9), 95.02 (C7), 98.44 (C5), 114.70 (C12), 114.85 (C15), 117.90 (C16), 130.56 (C11), 144.39 (C14), 155.72 (C6), 156.19 (C10), 156.48 (C8). ¹H NMR: δ ppm. (DMSO-d6) 9.089 (1H, s, OH), 8.873 (1H,s, OH), 8.777 (1H, s, OH), 8.694 (1H, s, OH), 6.876 (1H, d, J=2 Hz, H2′), 6.646 (2H, s, H-5′, 6′), 5.876 (1H, d, J=2 Hz, H8), 5.700 (1H, d, J=2 Hz, H6), 4.718 (1H, s, OH), 4.640 (1H, d, J=4.5 Hz, H2), 3.987 (1H, d, J=4.5 Hz, H3), 2.663 (1H, dd, J=4.6, 6.3 Hz, H4b), 2.463 (1H,dd, J=4.6, 6.3 Hz, H4a). MS: [M+1]⁺=291 m/e. This compound has been identified as epicatechin. The IC₅₀ values of epicatechin against the COX-1 and COX-2 enzymes are 7 μg/mL and 20 μg/mL, respectively.

Example 6

[0095] HPLC Quantification of Active Extracts from Acacia catechu

[0096] The flavan content in the organic and aqueous extracts from Acacia catechu were quantified by High Pressure Liquid Chromatography (HPLC) using a PhotoDiode Array detector (HPLC/PDA) and a Luna C18 column (250 mm×4.6 mm). The flavans were eluted from the column using an acetonitrile gradient from 10% to 30% ACN over a period of 20 minutes, followed by 60% ACN for five minutes. The results are set forth in Table 4. The flavans were quantified based on retention time and PDA data using catechin and epicatechin as standards. The retention times for the two major flavans were 12.73 minutes and 15.76 minutes, respectively. The HPLC chromatograms are depicted in FIGS. 4A and 4B. TABLE 4 Free-B-Ring Flavonoid Content in Active Plant Extracts Weight of % Extractible % Flavans in Active Extracts Extract from BioMass Extract Acacia catechu 10.8 g 18.0% 0.998% (AE)* Acacia catechu 27.2 g 45.3% 30.37% (OE)*

Example 7

[0097] In vitro Study of COX Inhibitory Activity of Organic Extracts from Acacia catechu

[0098] In vitro efficacy and COX-2 specificity of extracts from Acacia catechu were tested in cell based systems for their ability to inhibit the generation of arachidonic acid metabolites. Cell lines HOSC, which constitutively express COX-2 and THP-1, which express COX-1 were tested for their ability to generate prostaglandin E2 (PGE2) in the presence of arachidonic acid.

[0099] COX-2 Cell Based Assay. HOSC (ATCC#8304-CRL) cells were cultured to 80-90% confluence. The cells were trypsinized, washed and resuspended in 10 mL at 1×10⁶ cells/mL in tissue culture media (MEM). The cell suspension (200 μL) was plated out in 96 well tissue culture plates and incubated for 2 hours at 37° C. and 5% CO₂. The media was then replaced with new HOSC media containing 1 ng/mL IL-1b and incubated overnight. The media was removed again and replaced with 190 mL HOSC media. Test compounds were then added in 10 μL of HOSC media and were incubated for 15 minutes at 37° C. Arachidonic acid in HOSC media (20 mL, 100 μM) was added and the mixture was incubated for 10 minutes on a shaker at room temperature. Supernatant (20 μL) was transferred to new plates containing 190 μL/well of 100 μM indomethacin in ELISA buffer. The supernatants were analyzed as described below by ELISA.

[0100] COX-1 Cell Based Assay. THP-1 cells were suspended to a volume of 30 mL (5×10⁵ cells/mL). TPA was added to a final concentration of 10 nM TPA and cultured for 48 hours to differentiate cells to macrophage (adherent). The cells were resuspended in HBSS (25 mL) and added to 96 well plates in 200 mL volume at 5×10⁵ cells/well. The test compounds in RPMI 1640 (10 μL) were then added and incubated for 15 minutes at 37° C. Arachidonic acid in RPMI (20 μL) was then added and the mixture was incubated for 10 minutes on a shaker at room temperature. Supernatant (20 μL) was added to Elisa buffer (190 μL) containing indomethacin (100 μM). The supernatants were then analyzed by ELISA, as described below.

[0101] COX-2 Whole Blood assay. Peripheral blood from normal, healthy donors was collected by venipuncture. Whole blood (500 μL) was incubated with test compounds and extracts for 15 minutes at 37° C. Lipopolysaccharide (from E. coli serotype 0111:B4) was added to a final concentration of 100 μg/mL and cultured overnight at 37° C. The blood was centrifuged (12,000×g) and the plasma was collected. Plasma (100 μL) was added to methanol (400 μL) to precipitate proteins. Supernatants were measured for PGE2 production by ELISA. This procedure is a modification of the methods described by Brideau et al. (1996) Inflamm. Res. 45:68-74.

[0102] COX-1 Whole Blood Assay. Fresh blood was collected in tubes not containing anti-coagulants and immediately aliquoted into 500 μL aliquots in siliconized microcentrifuge tubes. Test samples were added, vortexed and allowed to clot for 1 hour at 37° C. The samples were then centrifuged (12,000×g) and the plasma was collected. The plasma (100 μL) was added to methanol (400 μL) to precipitate proteins. Supernatants were measured for TXB2 production by ELISA. This procedure is a modification of the methods described by Brideau et al. (1996) Inflamm. Res. 45:68-74.

[0103] ELISA Assays. Immunolon-4 ELISA plates were coated with capture antibody 0.5-4 μg/mL in carbonate buffer (pH 9.2) overnight at 4° C. The plates were washed and incubated for 2 hours with blocking buffer (PBS+1% BSA) at room temperature. The plates were washed again and test sample (100 μL) was added and incubated for 1 hour at room temperature while shaking. Peroxidase conjugated secondary antibody was added in a 50 μL volume containing 0.5-4 mg/mL and incubated for 1 hour at room temperature while shaking. The plates were then washed three times and TMB substrate (100 μL) was added. The plates were allowed to develop for 30 minutes, after which the reaction was stopped by the addition of 1 M phosphoric acid (100 μL). The plates were then read at 450 nm using a Wallac Victor 2 plate reader.

[0104] Cytotoxicity. Cellular cytotoxicity was assessed using a colorimetric kit (Oxford biochemical research) that measures the release of lactate dehydrogenase in damaged cells. Assays were completed according to manufacturers' directions. Both purified flavans and standardized extract from Acacia catechu were tested. No cytotoxicity was observed for any of the tested compounds.

[0105] The results of the assays are set forth in Table 5. The data is presented as IC₅₀ values for direct comparison. With reference to Table 5, IC₅₀ values are generally lower for COX-1 than COX-2. Additionally, whole blood was also measured for the differential inhibition of PGE2 generation (a measure of COX-2 in this system) or thromboxane B2 (TXB2) (a measure of COX 1 activation). Referring to Table 5, these studies clearly demonstrate specificity for COX-2 inhibition within the assays based on whole blood cells. However, studies using the THP-1 and HOSC based model system actually showed greater selectivity for COX-1. Possible reasons for this discrepancy are the fundamental differences between immortalized cell lines that constitutively express each of the enzymes and primary cells derived from whole blood that that are induced to express COX enzymes. Primary cells are the more relevant model to study inflammation in vivo. Additionally, the compounds used to identify COX-1 vs. COX-2 activity vary in each of these systems and consequently are not directly comparable. TABLE 5 Inhibition of COX Activity in Whole Cell Systems Cell Line Based Assay Whole Blood Assay Plant Source IC₅₀ COX-2 IC₅₀ COX-1 IC₅₀ COX-2 IC₅₀ COX-1 Acacia catechu 78 μg/mL 22 μg/mL 40 μg/mL >50 μg/mL

Example 8

[0106] Inhibition of 5 Lipoxygenase by the Organic Extracts from Acacia catechu

[0107] 5-Lipoxygenase assay. Human recombinant 5-lipoxygenase enzyme (Cayman) was diluted (1:50) in assay buffer (50 mM Tris-HCl, pH 7.5, 2 mM CaCl₂, 1 mM ATP). Equal volumes of sample (1:500 dilution) and diluted enzyme were incubated together while shaking for 15 minutes. Substrate (umbelliferyl arachidonate, 1:5000 in assay buffer) was added and the mixture was incubated for 30 minutes at room temperature while shaking. The resulting luminescence was read using a Wallac Victor 2 plate reader. The results are set forth in FIG. 5.

Example 9

[0108] In vivo Study of COX Inhibitory Activity of Organic Extracts from Acacia catechu

[0109] In vivo inhibition of inflammation was measured using two model systems. The first system (ear swelling model) measures inflammation induced directly by arachidonic acid. This is an excellent measure of COX-2 inhibition, but does not measure any of the cellular events which occur upstream of arachidonic acid liberation from cell membrane phospholipids by phospholipase A2 (PLA2). Therefore, to determine how inhibitors function in a more biologically relevant response the air pouch model was employed. This model utilizes a strong activator of complement to induce an inflammatory response that is characterized by a strong cellular infiltrate and inflammatory mediator production including cytokines as well as arachidonic acid metabolites.

[0110] Ear Swelling Model. The ear swelling model is a direct measure of the inhibition of arachidonic acid metabolism. Briefly, arachidonic acid in acetone is applied topically to the ears of mice. The metabolism of arachidonic acid results in the production of proinflammatory mediators produced by the action of enzymes such as COX-2. Inhibition of the swelling is a direct measure of the inhibition of the enzymes involved in this pathway. The results are set forth in FIG. 6, which shows the effects of the extracts delivered either orally by gavage or interperitoneally (IP) at two time points (24 hours and 1 hour). Extracts from Acacia inhibited swelling when delivered by both IP and gavage (FIG. 6A and FIG. 6B).

[0111] Air Pouch Model. Because Acacia organic extracts were efficacious in the ear swelling model a standardized Acacia extract was also examined using the air pouch model of inflammation. Briefly, an air pouch was created on the back of mice by injecting 3 mL of sterile air. The air pouch was maintained by additional injections of 1 mL of sterile air every other day for a period of one week. Animals were dosed using the same methods and concentrations described for the ear-swelling model and injected with Zymosan (into the air pouch) to initiate the inflammatory response. After four hours, the fluid within the pouch was collected and measured for the infiltration of inflammatory cells, myeloperoxidase (MPO) activity (a measure of cellular activation, degranulation). The results are set forth in FIG. 7.

[0112]FIG. 7A shows the total number of cells collected from the air pouch fluid. While there was a strong response that was inhibited by controls (indomethacin), the standardized Acacia extract did not inhibit the infiltration of the inflammatory cells (chemotaxsis). Even though the chemotactic response was not diminished, the fluid was examined to determine whether the infiltrating cells have become activated by measuring MPO activity. FIGS. 7B and 7C demonstrate that MPO activity is significantly reduced when the extract is administered IP, but not by gavage. These data suggest that although the extracts do not inhibit the chemotactic response induced by complement activation, they are still effective at reducing inflammation through the prevention of release and production of pro-inflammatory mediators.

[0113] Arachidonic Acid induced ear swelling. The ability of Acacia extracts to directly inhibit inflammation in vivo was measured as previously described. (Greenspan et al. (1999) J. Med. Chem. 42:164-172; Young et al. (1984) J. Invest. Dermat. 82:367-371). Briefly, groups of 5 Balb/C mice were given dosages of test compounds either interperitoneally (I.P.) or orally by gavage, 24 hours and 1 hour prior to the application of arachidonic acid (AA). AA in acetone (2 mg/15 μL) was applied to the left ear, and acetone (15 μL) as a negative control was applied to the right ear. After 1 hour the animals were sacrificed by CO₂ inhalation and the thickness of the ears was measured using an engineer's micrometer. Controls included animals given AA, but not treated with anti-inflammatory agents and animals treated with AA and indomethacin (I.P.) at 5 mg/kg.

[0114] Air pouch model of inflammation. Air pouch models were adapted from the methods of Rioja et al. (2000) Eur. J. Pharm. 397:207-217. Air pouches were established in groups of 5 Balb/C mice by injection of sterile air (3 mL) and maintained by additional injections of 1 mL every 2 days for a period of six days. Animals were given dosages of test compounds either I.P. or orally by gavage, 24 hours and 1 hour prior to the injection of 1% Zymosan (1 mL) into the pouch. After 4 hours, the animals were sacrificed by CO₂ inhalation and the air pouches were lavaged with sterile saline(3 mL). The lavage fluid was centrifuged and the total number of infiltrating cells determined. Supernatants were also retained and analyzed for myleoperoxidase (MPO) activity and the presence of TNF-α by ELISA as measures of activation.

Example 10

[0115] Development a Standardized Extract from Acacia catechu

[0116]Acacia catechu (500 mg of ground bark) was extracted with the following solvent systems. (1) 100% water, (2) 80:20 water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5) 20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, (8) 60:40 methanol:THF. The extract was concentrated and dried under low vacuum. Identification of the chemical components was carried out by High Pressure Liquid Chromatography using a PhotoDiode Array detector (HPLC/PDA) and a 250 mm×4.6 mm C18 column. The chemical components were quantified based on retention time and PDA data using catechin and epicatechin as standards. The results are set forth in Table 6 and FIG. 8. As shown in Table 6 and FIG. 8, the flavan extract generated from solvent extraction with 80% methanol/water provided the best concentration of flavan components. TABLE 6 Solvents for Generating Standardized Flavan Extracts from Acacia catechu Extraction Weight of % Extractible Total amount % Catechins Solvent Extract from BioMass of Catechins in Extract 100% water 292.8 mg 58.56% 0.13 mg 12.02% water:methanol 282.9 mg 56.58% 0.13 mg 11.19% (80:20) water:methanol 287.6 mg 57.52% 0.15 mg 13.54% (60:40) water:methanol 264.8 mg 52.96% 0.19 mg 13.70% (40:60) water:methanol 222.8 mg 44.56% 0.15 mg 14.83% (20:80) 100% methanol 215.0 mg 43.00% 0.15 mg 12.73% methanol:THF 264.4 mg 52.88% 0.11 mg 8.81% (80:20) methanol:THF 259.9 mg 51.98% 0.15 mg 9.05% (60:40) 

1. A method for the simultaneous dual inhibition of the cyclooxygenase COX-2 and 5-lipoxygenase (5-LO) enzymes comprising administering a composition comprising a flavan or a mixture of flavans isolated from a plant selected from the Acacia genus of plants.
 2. The method of claim 1 wherein said flavan or mixture of flavans is selected from the group of compounds having the following structure:

wherein R₁, R₂, R₃, R₄ and R₅ are independently selected from the group consisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters of substitution groups, independently selected from the group consisting of gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; a carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized flavans; wherein R is an alkyl group having between 1-10 carbon atoms and X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate.
 3. The method of claim 1 wherein said plant is selected from the group consisting of the Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.
 4. The method of claim 1 wherein said flavan is isolated from a plant part.
 5. The method of claim 4 wherein the plant part is selected from the group consisting of stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts.
 6. A method for simultaneously preventing and treating COX-2 and 5-LO mediated diseases and conditions comprising administering to a host in need thereof an effective amount of a composition comprising a flavan or a mixture of flavans isolated from a plant selected from the Acacia genus of plants and a pharmaceutically acceptable carrier.
 7. The method of claim 6 wherein said flavan or mixture of flavans is selected from the group of compounds having the following structure:

wherein R₁, R₂, R₃, R₄ and R₅ are independently selected from the group consisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters of substitution groups, independently selected from the group consisting of gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; a carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized flavans; wherein R is an alkyl group having between 1-10 carbon atoms; and X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate.
 8. The method of claim 6 wherein said plant is selected from the group consisting of the Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.
 9. The method of claim 6 wherein said flavan is isolated from a plant part.
 10. The method of claim 9 wherein the plant part is selected from the group consisting of stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts.
 11. The method of claim 6 wherein the COX-2 and 5-LO mediated disease or condition is selected from the group consisting of inflammation associated with osteoarthritis, rheumatoid arthritis, menstrual cramps, Systemic Lupus Erythromatosis, psoriasis, chronic tension headache, migraine headaches, inflammatory bowl disease; topical wound and minor inflammation conditions selected from the group consisting of minor abrasions, sunburn or contact dermatitis; and solid cancers.
 12. The method of claim 6 wherein the flavan composition is comprised of 0.01% to 100% of the flavan.
 13. The method of claim 6 wherein the composition is administered in a dosage selected from 0.01 to 200 mg/kg of body weight.
 14. The method of claim 6 wherein the routes of the administration are selected from the group consisting of oral, topical, suppository, intravenous, and intradermic, intragaster, intramusclar, intraperitoneal and intravenous administration in an appropriate pharmaceutical formula.
 15. A method for isolating and purifying a composition of flavans having dual specificity for COX-2 and 5-LO from the Acacia genus of plants comprising: a) extracting the ground biomass of a plant selected from the Acacia genus of plants b) neutralizing and concentrating said extract; and c) purifying said neutralized and concentrated extract.
 16. The method of claim 15 wherein the composition of flavans is extracted with a solvent selected from the group consisting of water, alcohol, an organic solvent and/or a combination thereof.
 17. The method of claim 15 wherein the extract is purified by using a method selected from the group consisting of recrystallization, solvent partition, precipitation, sublimation, and/or chromatographic separation.
 18. The method of claim 18 wherein said chromatographic separation is selected from the group consisting of methods including, but not limited to, ion exchange chromatography, absorption chromatography, reverse phase chromatography, size exclusive chromatography, ultra-filtration or a combination of two or more of those methods. 